The Translation of Capped mRNAs Has an Absolute Requirement for the Central Domain of eIF4G but Not for the Cap-binding Initiation Factor eIF4E

Ali, Iraj K.; Jackson, Richard J.

doi:10.1101/sqb.2001.66.377

Cited by 6 publications

(3 citation statements)

References 39 publications

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“…These results with eIF4G p50 prompted us to examine whether similar changes occurred in the presence of the complete (native) eIF4F complex, in view of the fact that it is eIF4F that drives poliovirus RNA translation in the early stages of infection until sufficient viral 2A protease has accumulated to promote cleavage of all the eIF4G component into an N-terminal one-third fragment (with the eIF4E interaction site) and a C-terminal two-thirds (p100) fragment; p100 is thought to have similar IRES-binding properties as the p50 central domain and similar activity in promoting picornavirus IRES functionality, at least with the EMCV IRES, although it appears slightly more potent than p50 (Ali and Jackson, 2001). The presence of native eIF4F resulted in qualitatively the same changes in the pattern of cleavages generated by the Fe(II) PTB 0 derivatives as were seen with the eIF4G p50 domain (compare Figure 5 and Supplementary Figure S2): a strong decrease in cleavage intensity at nt 537 (D284C), with a lesser decrease at nt 473 (D284C), and increased cleavage band intensity at nt 453 (D242C).…”

Section: Ptb and The Central Domain Of Eif4g Reciprocally Cause Sligh...mentioning

confidence: 99%

Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding

Kafasla

Morgner

Robinson

et al. 2010

EMBO J

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Tethered hydroxyl-radical probing has been used to determine the orientation of binding of polypyrimidine tractbinding protein (PTB) to the poliovirus type 1 (Mahoney) (PV-1(M)) internal ribosome entry site/segment (IRES)-the question of which RNA-binding domain (RBD) binds to which sites on the IRES. The results show that under conditions in which PTB strongly stimulates IRES activity, a single PTB is binding to the IRES, a finding which was confirmed by mass spectrometry of PTB/IRES complexes. RBDs1 and 2 interact with the basal part of the Domain V irregular stem loop, very close to the binding site of eIF4G, and RBDs3 and 4 interact with the single-stranded regions flanking Domain V. The binding of PTB is subtly altered in the presence of the central domain (p50) of eIF4G, and p50 binding is likewise modified if PTB is present. This suggests that PTB stimulates PV-1(M) IRES activity by inducing eIF4G to bind in the optimal position and orientation to promote internal ribosome entry, which, in PV-1(M), is at an AUG triplet 30 nt downstream of the base of Domain V.

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Section: Ptb and The Central Domain Of Eif4g Reciprocally Cause Sligh...mentioning

confidence: 99%

Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding

Kafasla

Morgner

Robinson

et al. 2010

EMBO J

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“…3B), but it can be greatly stimulated by addition of either the C-terminal two-thirds fragment (p100) or the central one-third (p50) of eIF4G (De Gregorio et al 1998;Ali et al 2001;Ali and Jackson 2002). Figure 7A shows that although these conditions, especially with p50, give rise to leaky scanning on the TPI/norm control and, to a lesser extent, on TPI/+8norm mRNA, it is nevertheless clear that p100 and p50 promoted even more efficient reinitiation on these uncapped mRNAs than is seen with capped or uncapped TPI/ter mRNAs in the standard lysate system (Figs.…”

Section: The Central Domain Of Eif4g Can Promote Reinitiationmentioning

confidence: 99%

“…The uncapped (CAA) 19 /2+8ter and its matched control, (CAA) 19 /+8norm, were translated at 100 mM added KCl, either in standard lysate, or in the eIF4G-depleted system (∼95% depletion) we have described recently (Ali et al 2001;Ali and Jackson 2002), which was either supplemented directly with recombinant p100 or p50 (Fig. 8A), or first preincubated with R362→ Q dominant negative eIF4A (to ensure that any residual traces of eIF4F, or 4G, would be completely inactivated) and then supplemented with p50 or p100 (Fig.…”

Section: Reinitiation Is Impaired If the Primary Scanning-dependent Imentioning

confidence: 99%

What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?

Pöyry¹,

Kaminski²,

Jackson³

2003

Genes Dev.

165

197

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If the 5-proximal AUG triplet in a mammalian mRNA is followed by a short open reading frame (sORF), a significant fraction of ribosomes resume scanning after termination of sORF translation, and reinitiate at a downstream AUG. To examine the underlying mechanism, we examined reinitiation in vitro using a series of mRNAs that differed only in the 5-untranslated region (UTR). Efficient reinitiation was found to occur only if the eIF4F complex, or at a minimum the central one-third fragment of eIF4G, participated in the primary initiation event at the sORF initiation codon. It did not occur, however, when sORF translation was driven by the classical swine fever virus or cricket paralysis virus internal ribosome entry sites (IRESs), which do not use eIF4A, 4B, 4E, or 4G. A critical test was provided by an mRNA with an unstructured 5-UTR, which is translated by scanning but does not absolutely need eIF4G and eIF4A: There was efficient reinitiation in a standard reticulocyte lysate, when initiation would be largely driven by eIF4F, but no reinitiation in an eIF4G-depleted lysate. These results suggest that resumption of scanning may depend on the interaction between eIF4F (or the eIF4G central domain) and the ribosome being maintained while the ribosome translates the sORF.[Keywords: Scanning ribosome mechanism; initiation factor eIF4F; initiation factor eIF4G; cricket paralysis virus IRES; classical swine fever virus (pestivirus) IRES] Supplemental material is available at http://www.genesdev.org.

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Alternative mechanisms of initiating translation of mammalian mRNAs

Jackson

2005

Biochemical Society Transactions

Self Cite

129

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Of all the steps in mRNA translation, initiation is the one that differs most radically between prokaryotes and eukaryotes. Not only is there no equivalent of the prokaryotic ShineDalgarno rRNA-mRNA interaction, but also what requires only three initiation factor proteins (aggregate size ∼125 kDa) in eubacteria needs at least 28 different polypeptides (aggregate >1600 kDa) in mammalian cells, which is actually larger than the size of the 40 S ribosomal subunit. Translation of the overwhelming majority of mammalian mRNAs occurs by a scanning mechanism, in which the 40 S ribosomal subunit, primed for initiation by the binding of several initiation factors including the eIF2 (eukaryotic initiation factor 2)-GTP-MettRNA i complex, is loaded on the mRNA immediately downstream of the 5 -cap, and then scans the RNA in the 5 → 3 direction. On recognition of (usually) the first AUG triplet via base-pairing with the Met-tRNA i anticodon, scanning ceases, triggering GTP hydrolysis and release of eIF2-GDP. Finally, ribosomal subunit joining and the release of the other initiation factors completes the initiation process. This sketchy outline conceals the fact that the exact mechanism of scanning and the precise roles of the initiation factors remain enigmatic. However, the factor requirements for initiation site selection on some viral IRESs (internal ribosome entry sites/segments) are simpler, and investigations into these IRES-dependent mechanisms (particularly picornavirus, hepatitis C virus and insect dicistrovirus IRESs) have significantly enhanced our understanding of the standard scanning mechanism. This article surveys the various alternative mechanisms of initiation site selection on mammalian (and other eukaryotic) cellular and viral mRNAs, starting from the simplest (in terms of initiation factor requirements) and working towards the most complex, which paradoxically happens to be the reverse order of their discovery. Historical perspectiveAlthough it was mammalian systems that provided the first insights, some 50 years ago, into the mechanism of protein biosynthesis and the role of ribosomes, this was soon overhauled by research into bacterial mRNA translation and thus further investigation into mammalian systems became rather a 'compare and contrast' exercise. Throughout the 1960s, when the emphasis was mainly on deciphering the genetic code and studying mechanisms of elongation, it seemed that eubacterial and eukaryotic protein synthesis were rather similar processes, and the discovery that both systems used an AUG codon and a dedicated initiator Met-tRNA for initiation raised false hopes that initiation, too, might be similar.However, by the mid 1970s, it was becoming clear that, in most other respects, initiation in the two systems must be very different. Sequencing the ends of rRNAs revealed that, although the 3 -ends of prokaryotic 16 S rRNA and eukaryotic 18 S rRNA are quite similar, the prokaryotic 16 S rRNA CCUCC motif, which is the core of the ShineDalgarno interaction, is precisely deleted from all euk...

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The Translation of Capped mRNAs Has an Absolute Requirement for the Central Domain of eIF4G but Not for the Cap-binding Initiation Factor eIF4E

Cited by 6 publications

References 39 publications

Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding

Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding

What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?

Alternative mechanisms of initiating translation of mammalian mRNAs

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